Anti-Mouse Ly-6G [Clone 1A8] — Purified in vivo PLATINUM™ Functional Grade

Clone 1A8 is extensively used in in vivo mouse studies for neutrophil depletion, serving as a crucial research tool for investigating the role of neutrophils in various disease models and biological processes.

Primary Application: Neutrophil Depletion

The 1A8 antibody specifically targets mouse Ly6G, a glycosylphosphatidylinositol (GPI)-anchored protein expressed on granulocytes in bone marrow and peripheral blood. This specificity makes it highly effective for selective neutrophil depletion without cross-reactivity with Ly6C, unlike other clones such as RB6-8C5.

Dosage and Administration Protocol

For neutrophil depletion studies, researchers typically administer 7.5-20 mg/kg of the 1A8 antibody, with the specific dose depending on multiple factors including mouse strain, age, body weight, disease model, and study duration. A common protocol involves injecting 500 ?g of anti-Ly6G antibody every 48 hours, starting from a specific timepoint in the experimental timeline (such as day 5 post-injection in disease models).

Research Applications

The antibody has been utilized across diverse research contexts, including:

• Infectious disease studies to understand neutrophil roles in pathogen clearance
• Inflammation research to investigate neutrophil contributions to inflammatory processes
• Cancer research to examine neutrophil function in tumor environments

Enhanced Versions for Long-term Studies

To address limitations in extended research protocols, scientists have developed murinized versions of the 1A8 antibody. These chimeric antibodies (mouse IgG2a and mouse IgG2c anti-Ly6G) maintain the same specificity but offer several advantages:

• Increased half-life in vivo
• Reduced immunogenicity by preventing anti-rat antibody production
• Improved efficacy in long-term studies
• Greater than 90% neutrophil depletion in peripheral blood

Experimental Considerations

Researchers must include appropriate isotype controls (rat IgG2a) to eliminate background effects and validate depletion specificity. Flow cytometry analysis is essential for measuring depletion efficiency, as the antibody may have limited efficacy against immature neutrophils. Studies have shown that approximately 28% of neutrophils may persist after treatment, particularly less mature cells with lower CD101 and CXCR2 expression and higher CXCR4 expression.

The 1A8 clone represents a fundamental tool in neutrophil biology research, enabling scientists to dissect the complex roles of these immune cells in health and disease through precise in vivo manipulation.

Commonly used antibodies or proteins employed alongside 1A8 (anti-Ly6G) in the literature typically target other leukocyte markers to enable comprehensive immune cell profiling, particularly in flow cytometry, immunohistochemistry, and depletion studies.

Frequently co-used antibodies or markers with 1A8 include:

• Anti-CD11b (M1/70): A marker for macrophages and myeloid cells; often combined with 1A8 for phenotypic analysis of myeloid cell populations.
• Anti-Ly6C: Used to distinguish monocytes and subsets of myeloid derived suppressor cells (MDSCs); important since Ly6C and Ly6G help differentiate between granulocyte and monocyte lineages.
• Anti-CD3e: T-cell marker, facilitates exclusion or analysis of T cells in leukocyte gating strategies.
• Anti-NK1.1: Natural killer (NK) cell marker, sometimes included in multi-color panels to analyze or exclude NK cells.
• Anti-B220/CD45R: B cell marker, often included for full leukocyte profiling.
• Anti-CD45: Pan-leukocyte marker, helps in the identification of total leukocyte populations.
• GR-1 (RB6-8C5): Binds both Ly6G and Ly6C, but is less specific than 1A8; sometimes used for comparative purposes.
• Isotype controls: Used to set specificity gates and control for background, especially with in vivo depletion antibodies like 1A8.

Summary Table of Common Antibodies Used with 1A8:

These markers are commonly used in multi-color panels to precisely define and distinguish immune cell subsets within complex samples.

Experimental context: In many studies, 1A8 is paired with these antibodies to specifically exclude or analyze neutrophils (Ly6G+), delineate monocytes versus granulocytes, and to account for all major leukocyte populations in tissue or blood samples. Isotype controls are always recommended to ensure specificity in both in vivo and in vitro applications.

Clone 1A8 represents a significant advancement in neutrophil research methodology, with key findings emerging from multiple studies that have utilized this antibody for specific neutrophil depletion. The antibody targets Ly-6G, a surface marker found exclusively on neutrophils, making it a highly specific tool for studying neutrophil function in vivo.

Specificity and Effectiveness

The 1A8 clone demonstrates remarkable specificity compared to earlier antibodies like Gr-1 (clone RB6-8C5), which depletes multiple cell types including neutrophils, monocytes, and certain CD8 T cell subsets. Studies have confirmed that the Ly-6G 1A8 antibody specifically depletes neutrophils without affecting other immune cell populations, providing researchers with a more precise tool for investigating neutrophil-specific functions.

Enhanced Depletion Strategies

A major breakthrough came with the development of a murinized version of the 1A8 antibody, which incorporates the same variable region as the original rat antibody but utilizes a mouse IgG2a isotype. This modification offers several critical advantages:

• Enhanced efficacy: The mouse IgG2a isotype can bind all murine Fc? receptors with optimal affinity profiles, enabling efficient antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP)
• Complement activation: The antibody can efficiently bind C1q, activating the complement pathway and inducing complement-dependent cytotoxicity (CDC)
• Extended duration: The murinized version achieves near-complete neutrophil depletion (greater than 90%) in peripheral blood for up to four weeks, and maintains effectiveness for up to six weeks in tumor-bearing mice

Novel Neutrophil Functions

Research using the 1A8 clone has revealed previously unknown roles for neutrophils in immune responses. In studies of bacterial infections, particularly with Listeria monocytogenes, neutrophil-specific depletion using 1A8 demonstrated that neutrophils contribute to bacterial clearance through TNF-? production rather than interferon-?. This finding challenges previous assumptions about neutrophil effector mechanisms and highlights the importance of using specific depletion methods.

Research Methodology Impact

The availability of the specific 1A8 antibody has significant implications for interpreting historical research. Studies that previously used the less-specific Gr-1 antibody may have attributed functions to neutrophils that were actually performed by other cell types, or conversely, may have missed neutrophil-specific contributions due to the confounding effects of depleting multiple cell populations simultaneously.

Tissue-Specific Applications

The murinized 1A8 antibody has proven effective across multiple mouse strains (C57Bl6/J, Balb/c, NXG, and SCID mice) and can successfully deplete neutrophils not only from peripheral blood but also from specific tissues, including tumor environments. This versatility makes it valuable for studying neutrophil functions in various pathological contexts, from infectious diseases to cancer research.

The collective findings from 1A8 citations represent a paradigm shift in neutrophil research, providing tools for more precise functional studies and revealing novel aspects of neutrophil biology that were previously obscured by less specific depletion methods.

Clone 1A8 (anti-Ly6G) is primarily used for neutrophil depletion in mice, with dosing regimens typically ranging from 100–250??g per mouse, administered intraperitoneally every 3 days or 3 times per week. However, the effective dose, frequency, and outcome can vary significantly across different mouse models and experimental settings.

Essential Context and Supporting Details

• Standard Regimen:

  • Dose: 100–250??g per mouse
  • Route: Intraperitoneal injection
  • Frequency: Every 3 days or 3 times per week
  • Application: Specific and selective depletion of neutrophils, mainly in tumor immunotherapy, infection, and inflammation models.

• Variation Based on Mouse Strain and Condition:

  • Efficiency Differences: 1A8-treated C57BL/6J and BALB/c mice show age- and condition-dependent efficacy. For example, no neutrophil depletion was observed in 24-week-old C57BL/6J mice or in the bone marrow of BALB/c mice in some studies. The same dose may be effective in younger mice or in other strains like FVB/N.
  • Model-Specific Adjustments: Mice often require higher doses of 1A8 compared to RB6-8C5 for comparable neutrophil depletion—a "2-fold higher dose of 1A8 than RB6-8C5" is typical.

• Duration of Depletion and Recovery:

  • Transient Effect: After a single treatment, neutrophil depletion is marked at 2 days, but neutrophil counts often recover rapidly thereafter in 1A8-treated mice.
  • For Long-Term Depletion: Repeat dosing is necessary; the schedule (e.g., every 3 days) is designed to maintain neutropenia.

• Special Considerations:

  • Reduced Efficacy in Certain Disease Models or Older Animals: In some infection models (e.g., PbNK65-infected C57BL/6) or with age, 1A8 efficacy declines; this may require adjunctive approaches such as secondary antibody administration or even switching to alternate depletion strategies.
  • Verification of Depletion: Efficacy should be verified experimentally due to variability in response across mouse strains, age groups, and pathologic conditions.

Summary Table: Typical 1A8 Dosing Regimen Variability

Key variables impacting 1A8 dosing regimens are mouse strain, age, disease model, and intended duration of neutrophil depletion. Regular monitoring of neutrophil counts is strongly recommended to validate protocol efficacy in each specific model.